Biomacromolecules 2008, 9, 783–788
783
Molecular Dynamics Studies of Side Chain Effect on the β-1,3-D-Glucan Triple Helix in Aqueous Solution Tadashi Okobira,† Kentaro Miyoshi,† Kazuya Uezu,*,† Kazuo Sakurai,† and Seiji Shinkai‡ Department of Chemical Processes and Environments, Faculty of Environmental Engineering, The University of Kitakyushu, 1-1 Hibikino Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan, and Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 744 Motooka Nishi-ku, Fukuoka, Fukuoka 819-0395, Japan Received May 10, 2007; Revised Manuscript Received October 10, 2007
β-1,3-D-Glucans have been isolated from fungi as right-handed 61 triple helices. They are categorized by the side chains bound to the main triple helix through β-(1f6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an waterinsoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the β-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a β-(1f6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 Å was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/ side chain hydrogen bonding in schizophyllan and the hypothetical β-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.
Introduction There has been a great deal of interest in recent years in the β-1,3-D-glucan series because they showed antitumor and antiAIDS viral activity in humans.1–3 Following rigorous purification, the repeating units of curdlan, lentinan, and schizophyllan have been identified with X-ray crystallographic data indicating that all three glucans form a right-handed triple helical complex under aqueous conditions.4–7 The difference in the β-1,3-Dglucan structures occurs primarily in the side chain. Curdlan, so named because a sample curdles on heating, is structurally the simplest member of the β-1,3-D-glucans with no glycosyl side chains.8,9 Schizophyllan10,11 (Figure 1) and lentinan12 have β-(1f6)-D-glycosyl side chains at every third and fifth main chain glucose unit, respectively. The structure of zymosan, another member of the glucan family, has not formally been identified although it is predicted to contain long side chains of irregular length and a differing glycosyl linkage to schizophyllan. Curdlan is slightly soluble in water when the molecular weight is relatively high (ca. >8000).13,14 Schizophyllan can be solvated in sufficient quantities to afford liquid crystals. Teramoto et al. reported an abnormal optical rotatory dispersion when the concentrated schizophyllan solution was cooled toward the freezing point of water.15 This phenomenon was described to be related to cooperative motion of the schizophyllan side chain and surrounding water molecules. The critical temperature required to induce this effect is highly solvent-dependent. It was * To whom correspondence should be addressed. E-mail address: uezu@ env.kitakyu-u.ac.jp. † The University of Kitakyushu. ‡ Kyushu University.
Figure 1. The repeating unit of a β-1,3-D-glucan (schizophyllan) and the definitions of the two dihedral angles and the bridge angle.
proposed that the motion of water molecules around the side chains is much more hindered due to hydrogen bonding between the glycosyl side chains and the water. Therefore, the hindered water molecules around the side chains seem to be a key factor to understand and predict the properties of the glucan family in aqueous conditions. Our recent research reported on the formation of stoichiometric complexes of schizophyllan with polynucleotides experimentally and computationally.16–18 The triple-helix complex is constructed from two glucan helices and a single polynucle-
10.1021/bm700511d CCC: $40.75 2008 American Chemical Society Published on Web 02/08/2008
784 Biomacromolecules, Vol. 9, No. 3, 2008
Okobira et al.
Figure 2. The structures of the CUR crystal and solvated β-1,3-Dglucans. Comparison between (a) CUR27 (b) 6/1G-SPG, and (c) SPG. Each single main chain consists of 19 glucose units. SPG and 6/1GSPG have one side chain per three main chain glucose residues and one side chain per six main chain glucose residues, respectively.
otide helix and stabilized by hydrogen bonding between the main-chain glucose residues of glucan and the base moieties of polynucleotide. The major factor about the complexation is thought to be similarity of higher-order structure of β-1,3-Dglucan and polynucleotides. This polysaccharide/polynucleotide complexation was observed only in the β-1,3-D-glucan case, other polysaccharides were not complexed. In a word, it can be expected that a triple helix structure of β-1,3-D-glucan is important in the complexation with the polynucleotides. This complex has been reported to dissociate to its respective single helices at certain temperatures and salt concentrations.19 Smith degredation was employed for the systematic cleavage of side chains from the original schizophyllan. We observed a considerable improvement in the thermal stability of the triple helix as the side chains were removed to some extent; the melting point (Tm) was improved from 33.3 to 47.3 °C.20 The formation process of β-1,3-D-glucan/polynucleotides complex is strongly affected by the behavior of the side chains. In a previous publication, we clarified the hydrogen bonding types in the crystal structure of curdlan and β-(1f3)-xylan, which is composed of xylose units, by using semiempirical molecular orbital methods (MOPAC).21,22 Both polysaccharides have three types of hydrogen bonding in the crystal structure: intermolecular hydrogen bonding formed between the different chain at the same x-y plane (the hexagonal hydrogen bonding),23 intramolecular hydrogen bonding formed between the adjacent O2 atoms in the same chain (the right-handed helical hydrogen bonding),24,25 and intermolecular hydrogen bonding formed between different chains at the different x-y plane (the left-handed helical hydrogen bonding).21 These hydrogen bonding types seems to be characteristic in the β-1,3-D-glucan crystals. However, there is still debate on the degree of polymorphism.26 We also simulated the behavior of curdlan in aqueous solution at room temperature by the molecular dynamics method.27 The helical pitch of curdlan expanded by 20% with thermal motion compared to the crystal structure. The pitch is one of helix parameters and defined as the length between the six main chain glucose units along the helix c axis. In this paper, we simulated three kinds of β-1,3-D-glucan: curdlan (with no side chains, CUR); a hypothetical glucan with a side chain at every sixth glucose unit (6/1G-SPG); schizophyllan (with a side chain at every third glucose unit, SPG). And we investigated how water molecules interact to glucan side chains and affect the glucan structure in aqueous solution at room temperature.
Calculation Construction of Models. Three β-1,3-D-glucan models were investigated: curdlan (CUR), a hypothetical schizophyllan
Figure 3. Fluctuation of the helix pitches for CUR,27 6/1G-SPG, and SPG models during the simulation. The red line indicates the pitch for a CUR crystal structure determined by Deslandes et al.28 The pitch is defined as the length between the six main chain glucose units along the helix c axis.
Figure 4. Conformational energy map for a disaccharides. Contour lines of local minima and local maxima are plotted with 2.0 kcal/mol. Dihedral angles after 700 ps were indicated red (CUR crystal), black (CUR), green (6/1G-SPG), and blue (SPG).
derivative (6/1G-SPG), and schizophyllan (SPG). The helical main chain of all three models has 19 glucose units. The coordinates of the carbon and oxygen atom are the same as those in Deslandes’ CUR data28 determined by X-ray crystallography except for the hydrogen atomic coordinates in the second hydrogen group, and the hydrogen atoms were geometryoptimized using the semiempirical molecular-orbital method (AM1 method).21 The models of SPG and 6/1G-SPG were constructed by the following procedure. The hydrogen atoms of the C6-OH hydroxyl group in the CUR crystal main chain was deleted, a glucose residue was attached to the main chain so as to bond the C1 carbon of the side chain glucose and the C6-OH oxygen atom of the main chain, and the side chain structure was geometry-optimized using the Molecular Mechanics method. Since the glucose ring adopts the chair conformation at moderate temperatures, the conformation of the polysaccharide can be described by two dihedral and one bridge angles about the glycosidic bond. These parameters are defined as φ(O5-C1-O3′-C3′), ψ(C1-O3′-C3′-C4′), and τ(C1-O3′C3′) as illustrated in Figure 1. Crystallographic data were assigned to the parameters of CUR: φ ) –92°, ψ ) 126°, and τ ) 110.4° (in IUPAC, φ(HC1-C1-O3′-C3′) ) 29.1°, ψ(C1-O3′-C3′-HC3′) ) 9.6°). Computational Methods and Assumptions. Molecular dynamics (MD) calculations were carried out for all β-1,3-Dglucan models in aqueous conditions using periodic boundary conditions at NTP ensemble with Materials Explorer Version 2.0 Ultra (Fujitsu Ltd. Japan).29 The β-1,3-D-glucan models were
Side Chain Effects on Glucan Solubility
Biomacromolecules, Vol. 9, No. 3, 2008 785
Figure 5. The number of the hydrogen bonds between 500 and 700 ps: (a) the hexagonal intermolecular C2-OH hydrogen bonding; (b) the right handed helical intramolecular C2-OH hydrogen bonding; (c) the left handed helical intermolecular C2-OH hydrogen bonding; (d) intra hydrogen bonding between the C4-OH hydroxyl group and O5′ the ring oxygen; (e) intra hydrogen bonding between the C4-OH hydroxyl group and the C6-OH hydrogen group in the same residue.
placed at the center of a periodic box and solvated to a distance of 10 Å by 20000 water molecules. The box dimensions were 85 × 85 × 85 Å for all models. The size is large enough to be unaffected by the box boundary even for SPG. The AMBER force field30 with the GLYCAM93 parameter set31 for oligosaccharides was used throughout the study. For water models, the TIP3P force field32 was used. The initial structures of three β-1,3-D-glucans are all completely dry models at absolute zero temperature. The glucans were then immersed in water at 0 K and the MD calculations started. The integration time step (∆t) was set to 0.5 fs. The temperature and the pressure were controlled using the Nose´ method33,34 and the ParrinelloRahman method,35,36 respectively. The pressure was maintained at 1 atm. The temperature was ramped at 30 K per 10 ps from 0 K to a final temperature of 298 K. The MD system became thermally equilibrated at 298 K over 100 ps. The total calculation time was 700 ps. The energy map was created from Molecular Mechanics calculation of two β-1,3-linked glucose residues.25 Dihedral angles φ and ψ were changed at intervals of 10° and geometry optimized using the AMBER force field with the parm94 parameter set.37 The strain energy was calculated in vacuo.
The hydrogen bonding was defined based on the hydrogendonor distance and the angle made by covalent bonds to the donor and accepter atoms (distance less than 3.2 Å; angle less than 120°).
Results and Discussion Conformational Changes of the Helix due to the Side Chain. After 700 ps, the triple helix in all the three models was structurally stabilized with no sign of dissociation (Figure 2). Six glucose residues were contained in one turn of the helix for all models except for terminal glucose residues. The pitch is one of helix parameters and defined as the length between the six main-chain glucose units along the helix c axis. Figure 3 shows the average pitch of three strands during 600 ps. Initially, a rapid increase in pitch was observed due to the thermal motion in transition from 0 to 298 K. After approximately 500 ps, all the pitch reached an equilibrated state with minor fluctuation. The average pitch between 500 and 700 ps was 20.6 Å for CUR, 19.4 Å for 6/1G-SPG, and 18.8 Å for SPG. CUR stretched much more than 6/1G-SPG and SPG in aqueous solution (Figure 3). Even for SPG, the pitch was longer than that for dry-CUR; the thermal fluctuations could inhibit
786 Biomacromolecules, Vol. 9, No. 3, 2008
Okobira et al.
Figure 6. A snapshot of 6/1G-SPG (a) and SPG (b) in 700 ps. Blue and green circles highlight the water molecule bridged hydrogen bonding.
Figure 8. The top views of the triple helix structures for (a) CUR, (b) 6/1G-SPG, and (c) SPG in 700 ps.
Figure 7. The MSD of the bulk water and the water molecules around side chains for SPG (the water molecules within about 3 Å from the side chain). The MSD(t) was calculated in the last 200 ps trajectory.
the left-handed helical intermolecular hydrogen bonding between the C2-OH hydroxyl group and C2-OH hydroxyl group (hydrogen bonding are formed between different chains as the different x-y plane). Standard deviation σ of the pitch between 500 and 700 ps was 0.413 Å for CUR, 0.266 Å for 6/1G-SPG, and 0.173 Å for SPG. The pitch extension and thermal fluctuation were constrained as the side chains increased. Dihedral Angle Distribution of β-1,3-D-Glucans in Aqueous Solution. The strain energies for a series of conformations of disaccharide with varying dihedral angles were calculated in order to evaluate the dihedral angle change in the stable β-1,3D-glucan conformation. The conformational energy map for a disaccharide is shown in Figure 4. Since the bridge angle (τ) was not hardly changed, two dihedral angles (φ and ψ) of CUR (black circle), 6/1G-SPG (green circle), and SPG (blue circle) at 700 ps were indicated in Figure 4. The φ and ψ values of the CUR crystal structure are -92° and 126° (red circle). The φ values were distributed range of -156 to -69° for CUR, -114 to -58° for 6/1G-SPG, and -125 to -41° for SPG, respectively. The ψ values were distributed in a range of 68-180° for CUR, 85-171° for 6/1G-SPG, and 72-175° for SPG. The dihedral angle distribution of all models was very similar to the disaccharide model. The leaving from the
minimum energy conformation, observed in hydrated systems, is mainly due to the interaction with solvent and effect of thermal fluctuations. The dihedral angles of CUR were widely distributed compared to 6/1G-SPG and SPG. The 1,3-linkage of CUR was rotated far from the most stable conformation by thermal fluctuation, and the triple helix of CUR stretched to a large extent on the whole. The dihedral angles of the β-1,3-D-glucan in aqueous solution behaved like those of disaccharides. Hydrogen Bonding Inside the β-1,3-D-Glucan Triple Helix. Only three types of hydrogen bonding are possible inside the triple helix of the CUR crystal: the hexagonal intermolecular hydrogen bonding, the right-handed helical intramolecular hydrogen bonding, and the left-handed helical intermolecular hydrogen bonding. These hydrogen bonding types were observed in the solvated β-1,3-D-glucans (Figure 5). The righthanded helical intramolecular hydrogen bonding between the C2-OH hydroxyl group and C2-OH hydroxyl group (Figure 5b) was more frequently observed than other types (parts a and c of Figure 5). On the other hand, other two types of hydrogen bonding were observed in solvated β-1,3-D-glucans: between the C4-OH hydroxyl group and O5′ the ring oxygen (Figure 5d), and between the C4-OH hydroxyl group and the C6-OH hydroxyl group in the same residue (Figure 5e). For the SPG and 6/1GSPG, the former type of hydrogen bonding was frequently observed. These two types of hydrogen bonding are also observed in disaccharides (see Figure 4).38,39 Water versus Side Chain Interactions. CUR has many hydrogen bonds between the main chains and water molecules. 6/1G-SPG and SPG have additionally the side chain/main chains
Side Chain Effects on Glucan Solubility
Figure 9. (a) Incline angle of the models with and without side chains. (b) Snapshot of SPG after 700 ps. Only six residues are highlighted to improve visualization.
hydrogen bonding and the side chain/side chain hydrogen bonding via water molecules (Figure 6). The hydrogen bonding was defined based on the hydrogen-donor distance and the angle made by covalent bonds to the donor and accepter atoms (distance less than 3.2 Å, angle less than 120°). Circle A shows the side chain/main chains hydrogen bonding via one water molecule. Circle B shows the side chain/side chain hydrogen bonding through two water molecules. It was similar to the hydrogen bonding reported by Kony et al.40 Since the density of side chains is higher in SPG, the side chain/side chain hydrogen bonding via one water molecule (circle C) can be formed. These types of hydrogen bonding were continually observed in SPG. The side chain/side chain hydrogen bonding is contributed to restrain the extension of the glucan triple helix. It is reason why 6/1G-SPG and SPG were more compressed than CUR. The mean-square displacement (MSD) of the bulk water and the water molecules around side chains is shown in Figure 7. The motion of the water molecules around the side chains was obviously restricted compared to that of the bulk water. The side chain/side chain hydrogen bonding in Figure 6 was continually constructed by water molecules around side chains during about 4 ps. This phenomenon was observed intermittently. Teramoto et al. reported that the water molecules bound to SPG side chains undergo an order–disorder transition at a critical temperature above the freezing point of water.41,42 They postulated that the bound water molecule is involved in the hydrogen bonding between side chains. The water molecules shown in circle C of Figure 6 could be the bound water molecules. Formation of One-Dimensional Cavity. A top view of the three models at 700 ps is shown in Figure 8. For convenience, only six glucose residues (a full twist of the helix) are depicted. In CUR (Figure 8a), the center of the helix was occupied by the second hydroxyl atoms just like the CUR crystal. In contrast, both 6/1G-SPG (Figure 8b) and SPG (Figure 8c) exhibit cylindrical-shaped cavities of diameter about 3.5 Å. The main chain diameter at 700 ps was around 16.5 Å for all three models; there was no diameter expansion of the helix at the cavity formation. The angle between the glucose residues of the main chain and the helix c axis, here defined as the incline angle shown in Figure 9, was significantly changed in 6/1G-SPG and SPG. The incline angle in 6/1G-SPG and SPG maintained about 30° during 200 ps of the simulation while the angle in CUR was about 70° (Figure 10). Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/
Biomacromolecules, Vol. 9, No. 3, 2008 787
Figure 10. The variation of incline angles between 500 and 700 ps.
side chain hydrogen bonding in 6/1G-SPG and SPG, could cause the tilt of the main chain glucose residues to the helix.
Conclusion This paper has examined structural differences among three kinds of β-1,3-D-glucans with varying numbers of side chains in aqueous solution by molecular dynamics simulations. It was found that the high density of side chains in β-1,3-D-glucan causes the restraint of the helix extension by thermal fluctuation. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. A one-dimensional cavity was observed at the center of the helices for both 6/1G-SPG and SPG. This cavity size was about 3.5 Å; the side chain/main chain and the side chain/side chain hydrogen bonding in 6/1G-SPG and SPG could cause the tilt of the main chain glucose residues to the helix. These phenomena were caused by the effect of the side chain. This investigation will allow us to understand the behavior of water molecules and β-1,3-D-glucans in aqueous solution. Acknowledgment. We thank the SORST programme by the Japan Science and Technology Corporation (JST) and the PRESTO “Function and Organization” programme for their joint financial support.
References and Notes (1) Biopolymers, Polysaccharides II: Polysaccharides from Eukaryotes; Wiley-VCH:Weinheim, 2002; Vol. 6. (2) Di Luzio, N. R.; Williams, D. L.; McNamee, R. B.; Edwards, B. F.; Kitahara, A. Int. J. Cancer 1979, 24, 773–779. (3) Jagodzinski, P. P.; Wiaderkiewicz, R.; Kurzawski, G.; Kloczewiak, M.; Nakashima, H.; Hyjek, E.; Yamamoto, N.; Uryu, T.; Kaneko, Y.; Posner, M. R.; Kozbor, D. Virology 1994, 202, 735–745. (4) Bluhm, T. L.; Sarko, A. Can. J. Chem. 1977, 55, 293–299. (5) Takahashi, Y.; et al. internal reporting. Daitou Ltd., Japan. (6) Bluhm, T. L.; Deslandes, Y.; Marchessault, B. H. Carbohydr. Res. 1982, 100, 117–130. (7) Norisuye, T.; Yanaki, T.; Fujita, H. J. Polym. Sci., Polym. Phys. Ed. 1980, 18, 547–558. (8) Saito, H.; Misaki, A.; Harada, T. Agric. Biol. Chem. 1968, 32, 1261– 1269. (9) Harada, T.; Masada, M.; Fujimori, K.; Maeda, I. Agric. Biol. Chem. 1966, 30, 196–198. (10) Tabata, K.; Ito, W.; Kojima, T.; Kawabata, S.; Misaki, A. Carbohydr. Res. 1981, 89, 121–135. (11) Yanaki, K.; Ito, W.; Kojima, T.; Norisuye, T.; Takano, N.; Fujita, H. Biophys. Chem. 1983, 17, 337–342. (12) Ohno, N. Chem. Pharm. Bull. 1985, 33, 4522–4527.
788 Biomacromolecules, Vol. 9, No. 3, 2008 (13) Chan, T. W. D.; Chan, K. Y. Rapid Commun. Mass Spectrom. 2003, 17, 887–896. (14) Koumoto, K.; Umeda, M.; Numata, M.; Matsumoto, T.; Sakurai, K.; Kunitake, T.; Shinkai, S. Carbohydr. Res. 2004, 339, 161–167. (15) Teramoto, A. Prog. Polym. Phys. 1998, 41, 25–65. (16) Sakurai, K.; Shinkai, S. J. Am. Chem. Soc. 2000, 122, 4520–4521. (17) Sakurai, K.; Mizu, M.; Shinkai, S Biomacromolecules 2001, 2, 641– 650. (18) Miyoshi, K.; Uezu, K.; Sakurai, K.; Shinkai, S Biomacromolecules 2005, 6, 1540–1546. (19) Sakurai, K.; Iguchi, R.; Koumoto, K.; Kimura, T.; Hisaeda, Y.; Mizu, M.; Shinkai, S. Biopolymers 2002, 65, 1–9. (20) Koumoto, K.; Karinaga, R.; Mizu, M.; Anada, T.; Sakurai, K.; Kunitake, T.; Shinkai, S. Biopolymers 2004, 75, 403–411. (21) Miyoshi, K.; Uezu, K.; Sakurai, K.; Shinkai, S. Chem. BiodiVersity 2004, 1, 916–924. (22) Miyoshi, K.; Uezu, K.; Sakurai, K.; Shinkai, S. Carbohydr. Polym. 2006, 66, 352–356. (23) Atkins, E. D. T.; Parker, K. D. Nature 1968, 220, 784–785. (24) Sathyanarayana, B, K.; Rao, V, S, R. Biopolymers 1972, 11, 1379– 1394. (25) Frecer, V.; Rizzo, R.; Miertus, S. Biomacromolecules 2000, 1, 91– 99. (26) Pelosi, L.; Bulone, V.; Heux, L. Carbohydr. Polym. 2006, 66, 199– 207. (27) Miyoshi, K.; Uezu, K.; Sakurai, K.; Shinkai, S. Sen’i Gakkaishi 2006, 62, 251–257.
Okobira et al. (28) Deslandes, Y.; Marchessault, R. H.; Sarko, S. Macromolecules 1980, 13, 1466–1471. (29) Materials Explorer Ver 2.0 Ultra. Fujitsu Ltd., Japan. (30) Cornell, W. D. J. Am. Chem. Soc. 1995, 117, 5179–5197. (31) Woods, R. J.; Dwek, R. A.; Edge, C. J. J. Phys. Chem. 1995, 99, 3832–3846. (32) Jorgensen, W. L. J. Am. Chem. Soc. 1981, 103, 335–340. (33) Nose′, S. J. Chem. Phys. 1984, 81, 511–519. (34) Nose′, S. Mol. Phys. 1984, 52, 255–268. (35) Parrinello, M.; Rahman, A. Phys. ReV. Lett. 1980, 45, 1196–1199. (36) Parrinello, M.; Rahman, A. J. Appl. Phys. 1981, 52, 7182–7190. (37) Cornell, W. D.; Cieplak, P.; Layly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179–5197. (38) Palleschi, A.; Bocchinfuso, G.; Coviello, T.; Alhaique, F. Carbohydr. Res. 2005, 340, 2154–2162. (39) Kony, D. B.; Damm, W.; S, S.; Hünenberger, P. H. J. Phys. Chem. B 2004, 108, 5815–5826. (40) Kony, D. B.; Damm, W.; Serge, S.; van Gunsteren, W. F.; Hünenberger, P. H. Biophys. J. 2007, 93, 442–455. (41) Yoshiba, K.; Teramoto, A.; Nakamura, N. Biomacromolecules 2003, 4, 1348–1356. (42) Yoshiba, K.; Teramoto, A.; Nakamura, N. Biomacromolecules 2004, 5, 2137–2146.
BM700511D
